SARS-CoV-2 Spike (S) glycoprotein expression, purification and characterization in suspension human embryonic kidney cells 293

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dc.contributor.authorFREIRE, RENAN P.pt_BR
dc.contributor.authorSILVA, FELIPE D.pt_BR
dc.contributor.authorVITALE, PHELIPEpt_BR
dc.contributor.authorTODESCHINI, IRISpt_BR
dc.contributor.authorMENEZES, FILIPEpt_BR
dc.contributor.authorOLIVEIRA, JOAO E. dept_BR
dc.contributor.authorOLIVEIRA, MONA das N.pt_BR
dc.contributor.authorSOARES, CARLOS R.J.pt_BR
dc.coverageInternacionalpt_BR
dc.creator.eventoPROTEIN EXPRESSION IN ANIMAL CELLS CONFERENCE, 15thpt_BR
dc.date.accessioned2022-03-30T18:18:06Z
dc.date.available2022-03-30T18:18:06Z
dc.date.eventoSeptember 13-16, 2021pt_BR
dc.description.abstractSARS-CoV-2 is a zoonotic virus RNA positive, which became responsible to be the largest sanitary crisis faced by humanity: Coronavirus disease 2019 (COVID-19). Some symptoms include major sneeze conditions, who could evolve to severe acute respiratory syndrome, and in some cases, to death. Techniques for accurate detection of this virus are essential to promote an accurate diagnosis of infected patients. SARS-CoV-2 has several targets with clinical interest; although, the focuses is on Spike (S), a homotrimer glycoprotein, that interacts with angiotensin converting enzyme receptor (ACE2), developing the infection in host cells. Thus, we recognize that the demand for the glycoprotein S is necessary, requiring large amounts with high purity level. The current work has the main objective the transient expression of SARS-CoV-2 S protein into suspension human embryonic kidney cells 293 (HEK293), purification and characterization, to use it as a template for discovering new molecular markers. SARSCoV- 2 S modified protein cDNA was inserted into pαH plasmid, amplified, and purified. For transient recombinant protein expression, 7.5 x 107 HEK293 cells (Expi293FTM cells) was seeded in 27 mL Expi293™ culture Medium. The transfection was carried out with a cationic lipid ExpiFectamine™ and 30 μg of plasmid, mixed with 3 mL Opti-Mem® culture medium. Cell culture was maintained for seven days in 125 mL vent cap Erlenmeyer, 32 ºC, 8% CO2, under 125 rpm orbital shaker rotation. 10 mL aliquots were collected on four- and seven-day post transfection, stored at -80 ºC. Physical-chemical and biological characterizations were determined by SDS-PAGE, ELISA, and Western Blotting. Purification from 40 mL of conditioned medium was carried out in two steps: Strep-Tag affinity column, followed by a size exclusion Superose® 6 (10/300), 5.0 mg of oligomeric recombinant protein with 95% purity was obtained. We believe that this process can be easily adapted to different volumes, being very useful for obtaining, in a short time, enough pure and immunological active SARS-CoV-2 S for further studies and applications, such as, cryogenic electron microscopy, mass spectroscopy, N-glycan structures, antibody production and immunologic assays development.pt_BR
dc.event.siglaPEACept_BR
dc.identifier.citationFREIRE, RENAN P.; SILVA, FELIPE D.; VITALE, PHELIPE; TODESCHINI, IRIS; MENEZES, FILIPE; OLIVEIRA, JOAO E. de; OLIVEIRA, MONA das N.; SOARES, CARLOS R.J. SARS-CoV-2 Spike (S) glycoprotein expression, purification and characterization in suspension human embryonic kidney cells 293. In: PROTEIN EXPRESSION IN ANIMAL CELLS CONFERENCE, 15th, September 13-16, 2021, Online. <b>Abstract...</b> Disponível em: http://repositorio.ipen.br/handle/123456789/32906.
dc.identifier.orcid0000-0002-6937-1120pt_BR
dc.identifier.orcidhttps://orcid.org/0000-0002-7982-1789
dc.identifier.urihttp://repositorio.ipen.br/handle/123456789/32906
dc.local.eventoOnlinept_BR
dc.rightsopenAccesspt_BR
dc.subjectcoronaviruses
dc.subjectglycoproteins
dc.subjectkidneys
dc.subjectembryonic cells
dc.subjectdiagnostic techniques
dc.titleSARS-CoV-2 Spike (S) glycoprotein expression, purification and characterization in suspension human embryonic kidney cells 293pt_BR
dc.typeResumo de eventos científicospt_BR
dspace.entity.typePublication
ipen.autorFELIPE DOUGLAS DA SILVA
ipen.autorCARLOS ROBERTO JORGE SOARES
ipen.autorJOAO EZEQUIEL DE OLIVEIRA
ipen.autorFILIPE MENEZES BEZERRA
ipen.autorRENAN PASSOS FREIRE
ipen.codigoautor14230
ipen.codigoautor509
ipen.codigoautor425
ipen.codigoautor15315
ipen.codigoautor14408
ipen.contributor.ipenauthorFELIPE DOUGLAS DA SILVA
ipen.contributor.ipenauthorCARLOS ROBERTO JORGE SOARES
ipen.contributor.ipenauthorJOAO EZEQUIEL DE OLIVEIRA
ipen.contributor.ipenauthorFILIPE MENEZES BEZERRA
ipen.contributor.ipenauthorRENAN PASSOS FREIRE
ipen.date.recebimento22-03
ipen.event.datapadronizada2021pt_BR
ipen.identifier.ipendoc28628pt_BR
ipen.notas.internasAbstractpt_BR
ipen.type.genreResumo
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relation.isAuthorOfPublicationd6719ab2-f2e9-4d7c-9cea-a6316fc14c9e
relation.isAuthorOfPublicatione9e6346c-9b8b-4d39-8bdd-505ea6997646
relation.isAuthorOfPublicationec1e1f96-0b93-4c21-9f5e-b75733651fbc
relation.isAuthorOfPublication14a1e53d-57dc-41b7-956a-9173679f41db
relation.isAuthorOfPublication.latestForDiscovery14a1e53d-57dc-41b7-956a-9173679f41db
sigepi.autor.atividadeSOARES, CARLOS R.J.:509:810:Npt_BR
sigepi.autor.atividadeOLIVEIRA, JOAO E. de:425:810:Npt_BR
sigepi.autor.atividadeMENEZES, FILIPE:15315:810:Npt_BR
sigepi.autor.atividadeSILVA, FELIPE D.:14230:810:Npt_BR
sigepi.autor.atividadeFREIRE, RENAN P.:14408:810:Spt_BR
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